Electrophoresis is a process in which macromolecules are separated on the basis of their charge-to-mass ratios by forcing them to move through a gel by means of a voltage gradient applied across the gel. Those species having uniform charge-to-mass ratios, such as DNA and RNA, are sorted according to their sizes, since the smaller molecules are able to move through the gel matrix more rapidly.
There are two basic formats used routinely in apparatus for electrophoresis of DNA: vertical and horizontal units. The vertical units, in which the gels are cast between two vertical non-conductive plates, offer greater reproducibility because of the uniform gel configurations created by the plates. They also allow for the application of larger sample volumes due to the fact that the sample wells are cast in the same plane as the gels, while the wells in horizontal units are cast into the thickness of the gels. The horizontal units offer greater ease in casting the gels, and do not require the troublesome precautions against leaking associated with the vertical units.
The present device incorporates the advantages of the vertical unit with the ease of operation of the horizontal unit by the addition of a novel plate which forms an upper boundary on the surface of the gel slab. Other such top plates have been described for horizontal electrophoresis units (see, e.g., Elson, D., and Avital, S., U.S. Pat. No. 3,888,759), but they do not address the problems of restricted sample volumes. Their apparatus also requires a larger number of elements, making it relatively complicated to set up. In the present invention, the top plate serves not only to provide a smooth, uniform gel, it also provides a vertically oriented application well that allows for the same sample volumes normally associated with vertical apparatus. In addition, it is notably simpler in operation than that described by Elson and Avital.
There are several other advantages to the use of such a top plate, both in general and in respect to the use of the resultant gels in the subsequent blotting procedures. First, the uniform cross-section of the gel provides a more reproducible electrophoretic pattern. Second, the top plate serves as a thermal insulator so that when the agarose gels are cast, the rate of cooling is reduced, thus reducing convection as the gel sets. This also provides a more uniform gel. Third, gels only 0.15-0.3 mm thick can be cast reproducibly, and this allows for more rapid separation of DNA fragments and also more rapid transfer of these fragments during the subsequent blotting procedure. Fourth, the flat upper surface is desirable for the subsequent blotting procedure, in which a thin membrane is placed in contact with the gel surface.
The present invention also embodies a feature found previously only in vertical gel units: the ability to cast two-gel systems. These two-gel systems are used routinely in high-resolution procedures such as the well-known Laemmli technique (Laemmli, U. K., Nature, 1970). Such procedures offer the advantage of tighter, more concentrated bands in the electrophoresis gel, and hence a more rapid, higher resolution blot. The present invention uses a space-filling element or a blank in place of one portion of the top plate, causing the gel material to be excluded from a portion of the gel tray. After the gel has set, the space-filling element may be removed and another suitable gel material cast contiguously in the space so provided.
In the present device the tray in which the gels are cast performs an additional function. It is used to carry the gel from the electrophoresis unit to the blotting unit and the gel may be left on the tray throughout the blotting procedure. This greatly increases the ease with which the operator can handle the gels. It also allows for the use of much thinner gels, with the benefits common to such gels. In fact, this makes it possible to use thinner gels, and at lower gel concentrations, than is currently possible with either vertical or horizontal systems.
It is, therefore, an object of the current invention to provide a method and an apparatus for casting thin horizontal electrophoresis gels that shall have increased sample capacity.
It is another object of the present invention to provide a means of casting two-gel systems in a horizontal unit.
It is another object of the current invention to provide horizontal gels of uniform, well-defined cross sections and smooth, flat surfaces.
It is a further object of the present invention to provide a means by which gels of lower concentration and having thinner cross sections than is possible with present techniques may be cast, electrophoresed, and blotted conveniently.
A further object of the present invention is the provision of a novel method and apparatus for molding a thin gel slab and a contiguous high volume sample well simultaneously.
Other features and advantages of the present invention will become more apparent from an examination of the following specification when read in conjunction with the appended drawings, in which;